Typically, knockouts are made by homologously replacing a target gene with another sequence of choice, usually a reporter and a selection cassette, where the latter is preferably flanked by site-specific recombinase sites to empower removal of the selection cassette via the action of the cognate site-specific recombinase. The selection cassette can be subsequently removed either by treating cells with the corresponding cognate recombinase or by breeding mouse progeny to a “deletor” strain. For example, in the case of floxed alleles (where the sequence of interest is flanked by loxP sites), the cognate recombinase is Cre, and what remains in the genome is a single loxP site and the reporter.
A related strategy has been traditionally employed to generate conditional-null alleles. This involves flanking part of the gene of interest with site-specific recombinase recognition sites (such as lox for Cre, and FRT for Flp) in a manner such that upon action of the cognate recombinase, the region flanked by the site-specific recombinase recognition sites is deleted and the resulting allele is a null allele.
Although attempts have been made to incorporate both a null and a conditional functionality in one targeting vector and to accomplish building the corresponding modified alleles in a single targeting step, the methods that have resulted from such attempts have several drawbacks and have had mixed success. These drawbacks include, for example, lack of true functionality (i.e., the null version is not a true null, the conditional allele is not a true conditional, lack of reporter function, etc.) or inability to realize a practical working allele with the desired features.
Therefore, there is a need in the art for generating genetically modified organisms via targeting where the engineered loci are multifunctional loci, for example, a true KO-first allele and then a conditional-null or other conditional-mutant allele.